| Porcine reproductive and respiratory syndrome characterized by reprodutive failure in sows and respiratory disease in piglets, is caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRS was first reported in1996in China, which present was increasingly extensive and very dangerous in the pig industry. The rapid and specific diagnostic method has caused tremendous economic losses to the swine industry worldwide. The rapid, specific and sensitive diagnostic method was much more important to control this disease. The PRRSV is a small enveloped positive-strand RNA virus, which genome is about15kb in length and contains eight open reading frames. The N protein encoded by ORF7, is nueleocapsid protein, which is the highest content and the strongest immunogenicity in all of the virus protein, and it can firstly induce the specific non-neutralization antibodies after virus infection. Therefore, PRRSV N Protein has an important meaning in the early diagnosis and pathogenic and immune mechanism study. In this study, polymorphic genetic characterization of the N protein of PRRSV of mainland China in GenBank was analyzed. On this basis, the ORF7gene of China advantage popular strain was efficiently and solubly expressed using the prokaryotic expression system, and with the recombinant N protein as coating antigen, the indirect ELISA method for detection of antibodies against PRRSV was successfully established. The main research works were as following:1Genetic variation analysis of the N protein of PRRSV in mainland ChinaThe N protein of PRRSV in mainland China in GenBank was sequenced and analyzed. Results showed that two genotypes are recognized for all deposited256isolates of PRRSV in mainland China, the European type and the North American type. The majority of them belongs to the North American type, which can be divided into four different subtypes; The sequence alignment analysis indicated that there was the characteristic mutations among each subtypes of amino acid sites, and there was amino acid replacement existed epitopes of the N protein. Results suggested there were the genetic diversity among PRRSV N protein in China.2Prokaryotic expression of the N protein of China advantage popular strainAccording to ORF7gene sequence of China advantage popular strain, a pair of primers was designed and the ORF7gene sequence was amplified and cloned, then the recombinant plasmid pET-28a-N was constructed. The recombinant plasmid pET-28a-N was inserted into E.coli BL21(DE3) and induced expression with IPTG, and then expressed about the size of the22kDa of recombinant protein. Analyzed by Western blot, the fusion protein reacted with PRRSV-positive serum, and has satisfactory immunogenicity, which suggested that the PRRSV N protein obtained a valid expression in Prokaryotic expression system.3Establishment and application of the indirect ELISA diagnosis method for the N protein of PRRSVUsing purified N protein as coating antigen, optimizing the reaction conditions, an indirect ELISA diagonosis method for detection of antibodies against porcine reproductive and respiratory syndrome virus was established. Results showed that the coating concentration of antigen was2.23μg/mL and optimal dilution of serum was1:200, and the best response time of serum was incubated for1hour at37℃, and then the work reactive time of HRP-antibody and substrate color reagent were1hours at37℃and5minetes, respectively. The critical value between positive and negative was0.395. So the established indirect ELISA method has good specificity and reproducibility. The indirect ELISA method detected192clinical serum samples from different regions, compared to the IDEXX-ELISA detection kit. Results suggested that the established indirect ELISA method had better compliance than the IDEXX-ELISA detection kit, and had a high specificity and sensitivity. |
